POS1035 FOCAL ADHESION PROTEINS KINDLIN-1, -2 AND TALIN-1 ARE REGULATED IN IL-1Β-STIMULATED RHEUMATOID ARTHRITIS SYNOVIAL FIBROBLASTS
Authors: M. Arnold, S. Rehart, M. Sauerbier, C. Biehl, C. Heck, U. Müller-Ladner, E. Neumann
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Rheumatoid arthritis (RA) is a progressive chronic inflammatory autoimmune disease characterized by synovial hyperplasia, articular inflammation and excessive joint and bone erosion. Activated RA synovial fibroblasts (SF) are key players of cartilage erosion by attaching and invading into cartilage. The kindlin family of focal adhesion proteins consists of three members, whose function is to activate integrins and thereby enable focal adhesion. Integrins play a central role in regulating cell-matrix and cell-cell adhesion. The effects of integrin activation are cell adhesion and migration, which are known to be pathologically increased in RASF. It is well described that dysfunction of integrins and kindlins and their co-factor talin-1 are involved in the pathogenesis of certain diseases characterized by inflammation and malignant transformation. However, the role of kindlins and talin-1 in RA, and specifically RASF, remain unclear. <h3>Objectives</h3> To investigate the compartmental and cellular localisation of kindlin-1, -2, -3 and talin-1 in RA synovial tissue and RASF, and the impact of pro-inflammatory activation on their expression on mRNA and protein level. <h3>Methods</h3> Synovial fibroblasts were isolated from synovial tissue after joint replacement surgery (RA, osteoarthritis (OA)) or synovectomy of hand joints (non-arthritic controls). RASF were stimulated with 10 ng/ml IL-1β for 6, 17, 24 and 48 h. In a second approach, RASF were repetitively stimulated three times with 0.05 ng/ml IL-1β every 24 h. RNA was isolated, reverse-transcribed and qPCR performed for kindlin-1, -2, -3 and talin-1. Whole RNA sequencing was performed for the repetitive stimulation approach and evaluated using matrix combining pairwise contrasts (DESeq2 and DESeq) normalized counts and thresholds for significance of differential expression per contrast. Immunocytochemistry on RASF seeded on chamber slides was performed. Immunohistochemistry on synovial tissue was performed on 5 µm acetone-fixed cryosections. Biological replicates and Kruskal-Wallis-test with Dunn's multiple comparison were used for statistics. <h3>Results</h3> In RA synovial tissues, kindlin-2 and talin-1 protein were detectable adjacent to blood vessels, in the synovial lining layer and in the sublining. Kindlin-2 and talin-1 could be detected in cultured RASF with a consistent signal of the cell body without a nuclear signal. Kindlin-1 and -3 were neither detectable in synovial tissue nor in synovial fibroblasts. Relative mRNA levels (arbitrary units) of kindlins and talin-1 in RASF differed, with the strongest signal for talin-1 (663 ± 186), followed by kindlin-2 (88 ± 23), kindlin-1 (2.15 ± 2.06) and kindlin-3 (0.12 ± 0.08). In comparison to unstimulated controls, stimulation of RASF with IL-1β led to a temporary downregulation of kindlin-2 (-4.1-fold, p=0.0456) and talin-1 (-1.6-fold) after 17 h and returned to the baseline levels after 48 h. This time-dependent effect could also be observed by immunocytochemistry of IL-1β-stimulated RASF. Kindlin-1 mRNA expression was significantly upregulated after 6 h (2.9-fold, p=0.0178) of stimulation and downregulated after 17 h (-1.5-fold, p&lt;0.0001), 24 h (-0.7-fold, p=0.0004) and 48 h (-1.2-fold, p&lt;0.0001) compared to 6 h. After repetitive activation of RASF, kindlins and talin-1 were constantly regulated compared to the first stimulation (kindlin-1: 1.03-fold; kindlin-2: -1.06-fold; talin-1: 1.00-fold). In contrast, IL-6 showed an inflammatory adjustment to IL-1β leading to lower induction of IL-6 (49.1%) after repetitive IL-1β exposure. <h3>Conclusion</h3> Focal adhesion proteins kindlin-2 and talin-1, factors involved in integrin activation of cells, are present on RA synovial tissue and in RASF. As a time-dependent regulation of kindlin-2 and talin-1 after stimulation was observed, this effect may lead to a temporary and repetitive reproducible activation of RASF and activation of integrins, subsequently contributing to altered adhesion and migration of RASF under inflammatory arthritic conditions. <h3>REFERENCES:</h3> NIL. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> None Declared.